Recommendation
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Correspondence to
Dr Jan Damoiseaux, Central
Diagnostic Laboratory,
Maastricht University Medical
Center, Maastricht 6229 HX,The
Netherlands;
j​ an.​damoiseaux@​mumc.​nl
Received 13 September 2018
Accepted 23 January 2019
Published Online First
12 March 2019
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To cite: Damoiseaux J,
Andrade LEC, Carballo OG,
et al. Ann Rheum Dis
2019;78:879–889.
Clinical relevance of HEp-2 indirect
immunofluorescent patterns: the International
Consensus on ANA patterns (ICAP) perspective
Jan Damoiseaux,‍ ‍ 1 Luis Eduardo Coelho Andrade,2 Orlando Gabriel Carballo,3,4
Karsten Conrad,5 Paulo Luiz Carvalho Francescantonio,6 Marvin J Fritzler,7
Ignacio Garcia de la Torre,8 Manfred Herold,9 Werner Klotz,10
Wilson de Melo Cruvinel,11 Tsuneyo Mimori,12 Carlos von Muhlen,13 Minoru Satoh,14
Edward K Chan15
Abstract
The indirect immunofluorescence assay (IIFA) on HEp-
2 cells is widely used for detection of antinuclear
antibodies (ANA). The dichotomous outcome, negative
or positive, is integrated in diagnostic and classification
criteria for several systemic autoimmune diseases.
However, the HEp-2 IIFA test has much more to offer:
besides the titre or fluorescence intensity, it also provides
fluorescence pattern(s). The latter include the nucleus
and the cytoplasm of interphase cells as well as patterns
associated with mitotic cells. The International Consensus
on ANA Patterns (ICAP) initiative has previously reached
consensus on the nomenclature and definitions of HEp-2
IIFA patterns. In the current paper, the ICAP consensus
is presented on the clinical relevance of the 29 distinct
HEp-2 IIFA patterns. This clinical relevance is primarily
defined within the context of the suspected disease and
includes recommendations for follow-up testing. The
discussion includes how this information may benefit the
clinicians in daily practice and how the knowledge can
be used to further improve diagnostic and classification
criteria.
Introduction
Autoantibodies, as detected by the indirect immu-
nofluorescence assay (IIFA) on HEp-2 cells (IIFA
HEp-2), are recognised as important diagnostic
markers in a plethora of autoimmune diseases, in
particular the systemic autoimmune rheumatic
diseases (SARD).1 Although somewhat dated
by today’s standards, members of the American
College of Rheumatology (ACR) prepared an
evidence-based guideline for the usefulness of the
HEp-2 IIFA results for diagnostic and prognostic
purposes and also for meeting diagnostic criteria.2
That guideline was based on reactivity with nuclear
antigens as detected by IIFA on rodent tissue or
HEp-2 cells. More recently, the IIFA on HEp-2 cells
was reinforced as the gold standard for autoanti-
body screening in SARD.3
Interestingly, the HEp-2 IIFA test reveals much
more information than the mere absence or pres-
ence of autoantibodies, that is, the level of antibody
as well as the HEp-2 IIFA pattern. Based on titra-
tion or appropriate evaluation of the fluorescence
intensity, the antibody level can be determined and
this information has general concordance with the
Damoiseaux J, et al. Ann Rheum Dis 2019;78:879–889. doi:10.1136/annrheumdis-2018-214436
clinical relevance of the test result. Indeed, higher
antibody levels are better associated with SARD and
have an increased likelihood to identify the auto-
antigen in follow-up testing.4–6 The importance of
the level of autoantibodies is also recognised in the
ACR guideline as well as by the recommendations
issued by the European Autoimmunity Standardiza-
tion Initiative (EASI) and the International Union of
Immunologic Societies (IUIS) Autoantibody Stan-
dardization Subcommittee.2 7
The HEp-2 IIFA pattern may also reveal clini-
cally relevant information. This information is not
restricted to giving direction to follow-up testing for
antigen-specificity, but, for instance, the centromere
pattern is included in the classification criteria for
systemic sclerosis,8 while the nuclear dense fine
speckled pattern is reported to be more prevalent
in apparently healthy individuals as compared with
patients with SARD.9 To harmonise the names and
descriptions of the distinct HEp-2 IIFA patterns, an
ordered classification taxonomy was proposed.10
This proposal was subsequently elaborated on
by the International Consensus on ANA Patterns
(ICAP), initiated in parallel to the 12th Interna-
tional Workshop on Autoantibodies and Autoim-
munity (2014) held in Sao Paulo, Brazil. During this
workshop, a consensus was reached on the nomen-
clature and definitions of 28 HEp-2 IIFA patterns.
Each HEp-2 IIFA pattern was ascribed an alphanu-
meric code from AC-1 to AC-28.11 The consensus
nomenclature for each pattern and representative
images were also made available online at the ICAP
website (http://www.​ANApatterns.​org).
In addition to the nuclear patterns, important
cytoplasmic and mitotic patterns may also be
observed in HEp-2 IIFA analysis. Although
reporting non-nuclear patterns is considered clin-
ically relevant,7 for various jurisdictional reasons
there is no clear-cut consensus viewpoint on
reporting non-nuclear patterns as a negative or
positive test.12 With the understanding that the
term ‘Antinuclear antibody (ANA) test’ may be
inappropriate to designate a test that also addresses
autoantibodies to antigens in the cytoplasm and
mitotic apparatus, an alternative name, anticellular
antibodies, was suggested in the EASI/IUIS recom-
mendations.7 Recent publications from ICAP have
preferred the term HEp-2 IIFA as it covers the
   879
 Recommendation
whole spectrum of patterns that can be observed when using
the HEp-2 cells as substrate.13 14
Originally, the HEp-2 IIFA patterns were associated with
diseases, but it was anticipated that many of these associa-
tions are only valid if the antigen-specificity was confirmed by
follow-up testing. In subsequent ICAP workshops, it was agreed
that the disease associations should be replaced by clinical rele-
vance. In this current paper, we present the consensus on the
clinical relevance of the distinct HEp-2 IIFA patterns as achieved
by consecutive workshops and discussions among the executive
ICAP members.
Materials and methods
For discussion about the structure of clinical relevance templates
were prepared for AC-2 (LECA), AC-3 (JD) and AC-5 (MS).
This formed the basis of a guideline for description of each
AC pattern (EC). Of highest importance, it was agreed that the
information should be objective and helpful for the clinician, the
pattern–antigen associations should be put in the right clinical
context and information should be evidence-based.
In preparation for the third ICAP workshop in Kyoto (2016),
composition of the clinical relevance documents was started
for the nuclear patterns (JD, LECA, MS), cytoplasmic patterns
(CAvM, EKLC) and mitotic patterns (MH, TM). As far as already
available, the documents were commented on by the ICAP exec-
utive board and, after appropriate adjustment, discussed with
the workshop participants. The feedback from participants
mainly focused on the structure of the information provided,
on the required level of detail and the format of recommended
follow-up testing.
In anticipation of the fourth ICAP workshop in Dresden
(2017), the set of clinical relevance documents was completed
for all patterns. Further comments from the ICAP executive
board were included. The resulting documents were individ-
ually discussed with the workshop participants for nuclear
(JD), cytoplasmic (CAvM) and mitotic (MH) patterns. Besides
several substantive comments, there was general agreement that
the information should be provided in tabular format at two
distinct levels. The first level should contain information on
relevant follow-up testing in the respective clinical context, the
recommended follow-up tests should be commercially available
and detailed test characteristics should not be given because of
potential geographic and jurisdictional differences. Information
based on case reports or small patient cohorts, as well as infor-
mation on possible follow-up testing that is only available in
specialised research laboratories, should only be provided in the
second level information.
Tables for nuclear, cytoplasmic and mitotic patterns were
prepared for first and second level information (JD). These
tables were commented by the ICAP executive board and final-
ised by JD. Of note, since the starting point of the tables on
clinical relevance is the HEp-2 IIFA pattern and not the clinically
suspected disease, the tables do not list all autoantibodies related
to the respective disease.
Results
Nuclear HEp-2 IIFA patterns
To date, a total of 15 nuclear HEp-2 IIFA patterns have been
described, that is, AC-1–AC-14 and AC-29. Table 1 summarises
the clinical relevance of these patterns.8 9 14–79 Since AC-29 was
only recently described,14 the advice for follow-up testing for
autoantibodies to topoisomerase I (Scl-70) in case of clinical
suspicion of systemic sclerosis is also added as a note to the
880 Damoiseaux J, et al. Ann Rheum Dis 2019;78:879–889. doi:10.1136/annrheumdis-2018-214436
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clinical relevance of AC-1. In particular, disease-specific immu-
noassays, like autoimmune liver disease profile, inflammatory
myopathy profile, systemic sclerosis profile, are often only avail-
able in specialty clinical laboratories.
For six nuclear HEp-2 IIFA patterns (AC-3, 5, 7, 8, 12 and 13),
additional information about clinical relevance is summarised
in online supplementary table S1. Although some assays for
anti-CENP-A antibodies are commercially available, these anti-
bodies are included in online supplementary table S1 because the
majority of sera revealing the AC-3 pattern are also reactive with
CENP-B. In contrast to CENP-A, CENP-B is included in many
routine extractable nuclear antigens profiles.
Cytoplasmic HEp-2 IIFA patterns
Table 2 summarises the clinical relevance of the nine cyto-
plasmic HEp-2 IIFA patterns, that is, AC-15–AC-23.26 33 80–101
It is recognised that the distinction between AC-19 (dense fine
speckled) and AC-20 (fine speckled) can be challenging. More-
over, within the spectrum of anti-tRNA synthetase antibodies,
not all produce an HEp-2 IIFA pattern and only some anti-Jo-1
antibodies are considered to give the AC-20 pattern, while the
other anti-tRNA synthetase antibodies (EJ, KS, OJ, PL-7 and
PL-12) are more likely to reveal the AC-19 pattern. Solid infor-
mation on the pattern of two additional anti-tRNA synthetase
antibodies (Ha and Zo) is lacking. Overall, the relation between
these two cytoplasmic HEp-2 IIFA patterns and the distinct anti-
tRNA synthetase antibodies is subject to further discussion. In
clinical practice, the complete spectrum of the anti-tRNA synthe-
tase antibodies should be determined irrespective of the subtype
of cytoplasmic speckled pattern, that is, AC-19 or AC-20.
For seven cytoplasmic HEp-2 IIFA patterns (AC-15–AC-19,
AC-22 and AC-23), more detailed information is provided in
online supplementary table S2. In particular, for AC-16–AC-18,
the clinical associations are quite diverse, depending on the
antigen recognised. Overall, the clinical associations provided
are primarily based on antigen-specific immunoassays and not
on the HEp-2 IIFA pattern as such.
Mitotic HEp-2 IIFA patterns
The clinical relevance of the five mitotic patterns is summarised
in table 3,102–122 with more detailed information in online
supplementary table S3. As for the cytoplasmic patterns, clinical
associations for the mitotic patterns are primarily based on anti-
gen-specific immunoassays and not on the HEp-2 IIFA pattern
as such.
Discussion
In the current paper, we present the ICAP consensus on the clin-
ical relevance of 29 HEp-2 IIFA patterns defined by ICAP.11 14
The consensus on clinical relevance is defined in the clinical
context of the patient, that is, suspected disease, and includes
recommended follow-up testing within the spectrum of anti-
gen-specificities that are commercially available. Obviously, if
follow-up testing identifies the antigen, the clinical relevance can
be further refined.123
Defining the clinical relevance of HEp-2 IIFA patterns in the
context of disease manifestations is meant to be an important
tool for the clinician in the diagnostic work-up of patients
suspected of SARD. Unfortunately, good data on the associa-
tion between HEp-2 IIFA patterns and the distinct diseases are
lacking, probably due to reasons summarised below. There are
several reasons for not finding a perfect association between
HEp-2 IIFA patterns and diseases. First, pattern assignment in
 Recommendation

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Table 1 Nuclear HEp-2 IIFA patterns
Code AC pattern—clinical relevance Refs
AC-1 HOMOGENEOUS
►► Found in patients with SLE, chronic autoimmune hepatitis or juvenile idiopathic arthritis
►► If SLE is clinically suspected, it is recommended to perform a follow-up test for anti-dsDNA antibodies, 15, 16
alone or in combination with dsDNA/histone complexes (nucleosomes/chromatin); anti-dsDNA antibodies
are included in the classification criteria for SLE
►► If chronic autoimmune hepatitis or juvenile idiopathic arthritis is suspected, follow-up testing is not 17
recommended because the respective autoantigens revealing the AC-1 pattern are not completely defined
Notes: Although autoantibodies to Topoisomerase I (formerly Scl-70) may be reported as nuclear 14, 18
homogeneous, they typically reveal a composite AC-29 HEp-2 IIFA pattern; as such, clinical suspicion of SSc
may warrant follow-up testing for reactivity to this antigen.
Although AC-1 is the most prevalent pattern in chronic autoimmune hepatitis, other HEp-2 IIFA patterns may 19
occur, but also for these patterns the autoantigens are not completely defined.
AC-2 DENSE FINE SPECKLED
►► Commonly found as high titer HEp-2 IIFA-positive in apparently healthy individuals or in patients who do 9
not have a systemic autoimmune rheumatic disease (SARD)
►► The negative association with SARD is only valid if the autoreactivity is confirmed as being directed to 20, 21
DFS70 (also known as LEDGF/p75) and if no other common ENA is recognized
►► Both in apparently healthy individuals as well as patients who do not have a SARD the AC-2 pattern may 22
be caused by autoantibodies to other antigens than DFS70
Note: Confirmatory assays for anti-DFS70 antibodies may be available only in specialty clinical laboratories.
AC-3 CENTROMERE (see online supplementary table S1 for further details
►► Commonly found in patients with limited cutaneous SSc, and as such included in the classification criteria 8, 15, 23
for SSc
►► In combination with Raynaud phenomenon, the AC-3 pattern is prognostic for onset of limited cutaneous 15, 23
SSc
►► Strongly associated with antibodies to CENP-B; especially in case of low titers, confirmation by an antigen- 15
specific immunoassay is recommended to support the association with limited cutaneous SSc; the CENP-B
antigen is included in many routine ENA profiles
►► The AC-3 pattern is also apparent in a subset of patients with PBC; these patients often have both SSc as 15
well as PBC
AC-4 FINE SPECKLED
►► Present to a varying degree in distinct SARD, in particular SjS, SLE, subacute cutaneous lupus 15
erythematosus, neonatal lupus erythematosus, congenital heart block, DM, SSc, and SSc-AIM overlap
syndrome
►► If SjS, SLE, subacute cutaneous lupus erythematosus, neonatal lupus erythymatosus, or congenital heart 15
block is clinically suspected, it is recommended to perform follow-up tests for anti-SS-A/Ro (Ro60) and
anti-SS-B/La antibodies; in most laboratories these antigens are included in the routine ENA profile
►► Autoantibodies to SS-A/Ro are part of the classification criteria for SjS (the criteria do not distinguish 25
between Ro60 and Ro52/TRIM21)
►► If SSc, AIM, or to a lesser extend SLE, is clinically suspected, it is recommended to perform follow-up 26
tests for detecting autoantibodies to Mi-2, TIF1γ, and Ku; these antigens are typically included in disease
specific immunoassays (i.e., inflammatory myopathy profile*)
►► Autoantibodies to Mi-2 and TIF1γ are associated with DM; autoantibodies to TIF1γ in patients with DM, 26, 27
although rare in the overall AC-4 pattern, is strongly associated with malignancy in old patients
►► Autoantibodies to Ku are associated with SSc-AIM and SLE-SSc-AIM overlap syndromes 26
Notes: Anti-SS-A/Ro (Ro60) and AIM-specific autoantibodies may be undetected in HEp-2 IIFA-screening. 28
AC-5 LARGE/COARSE SPECKLED (see online supplementary table S1 for further details)
►► Present to a varying degree in distinct SARD, in particular SLE, SSc, MCTD, SSc-AIM overlap syndrome, and 29
UCTD (i.e, patients with rheumatic symptoms without a definite SARD diagnosis)
►► If SLE is clinically suspected, it is recommended to perform follow-up tests for anti-Sm and anti-U1RNP 16, 30, 31
antibodies; these antigens are commonly included in the routine ENA profile; anti-Sm antibodies are
included in the classification criteria for SLE
►► If SSc is clinically suspected, it is recommended to perform a follow-up test for anti-RNApol III antibodies 8
(e.g., SSc profile*); the anti-RNApol III antibodies are included in the classification criteria for SSc
►► If MCTD is clinically suspected, it is recommended to perform a follow-up test for anti-U1RNP antibodies; 32
the antigen is commonly included in the routine ENA profile; anti-U1RNP antibodies are included in the
diagnostic criteria for MCTD
►► If the SSc-AIM overlap syndrome is clinically suspected, it is recommended to perform follow-up tests for 26, 33
anti-U1RNP and anti-Ku antibodies; these antigens are included in the routine ENA profile (U1RNP), or in
disease specific immunoassays (Ku, i.e., inflammatory myopathy profile* and SSc profile*)
►► In non-SARD individuals in the general population, the presence of the AC-5 pattern is not associated with
the autoantigens mentioned above and most often concerns low antibody titers
Continued

Damoiseaux J, et al. Ann Rheum Dis 2019;78:879–889. doi:10.1136/annrheumdis-2018-214436 881

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Table 1 Continued
Code AC pattern—clinical relevance Refs
AC-6 MULTIPLE NUCLEAR DOTS
►► Found in a broad spectrum of autoimmune diseases, including PBC, AIM (DM), as well as other 34
inflammatory conditions
►► If PBC is clinically suspected, it is recommended to perform follow-up tests for anti-Sp100 (and PML/ 17, 35, 36
Sp140) antibodies; in particular anti-Sp100 antibodies have the best clinical association with PBC and
have added value, especially when associated with AMA; the Sp100 (and PML-Sp140) antigen is included
in disease specific immunoassays (ie, liver profile*)
►► If DM is clinically suspected, it is recommended to perform a follow-up test for anti-MJ/NXP-2 antibodies; 37–39
these anti-MJ/NXP-2 antibodies are highly specific for AIM, are found in up to one third of patients with
juvenile DM, and have been reported to be associated with malignancies in adult AIM patients; the
antigen is included in disease specific immunoassays (i.e., inflammatory myopathy profile*)
AC-7 FEW NUCLEAR DOTS (see online supplementary table S1 for further details)
►► The AC-7 pattern has low positive predictive value for any disease 40, 41
►► Antigens primarily localized in the dots include p80-coilin and SMN complex; specific immunoassays for 42, 43
these autoantibodies are currently not commercially available
AC-8 HOMOGENEOUS NUCLEOLAR (see online supplementary table S1 for further details)
►► Found in patients with SSc, SSc-AIM overlap syndrome, and patients with clinical manifestations of other 44–46
SARD
►► If limited cutaneous SSc is clinically suspected, it is recommended to perform a follow-up test for anti-Th/ 44, 45
To antibodies; the antigen is included in disease specific immunoassays (ie, SSc profile*)
►► If SSc-AIM overlap syndrome is clinically suspected, it is recommended to perform a follow-up test for 46
anti-PM/Scl antibody reactivity; the antigen may be included in the routine ENA profile and is included in
disease specific immunoassays (i.e., inflammatory myopathy profile* and the SSc profile*); in general, anti-
PM/Scl antibodies yield a diffuse nuclear fine speckled staining in addition to the AC-8 pattern
►► Other antigens recognized include B23/nucleophosmin, No55/SC65, and C23/nucleolin, but the
clinical significance of these autoantibodies is not well established; specific immunoassays for these
autoantibodies are currently not commercially available
Notes: Although some anti-Th/To antibody immunoassays are commercially available, technical issues relating 44, 47
to the limited sensitivity of these immunoassays should be taken in to consideration.
AC-9 CLUMPY NUCLEOLAR
►► Found in patients with SSc 48
►► If SSc is clinically suspected, it is recommended to perform a follow-up test for anti-U3RNP/fibrillarin 48
antibodies; the antigen is included in disease specific immunoassays (i.e, SSc profile*)
►► If confirmed as anti-U3RNP/fibrillarin reactivity by immunoassay, the clinical association is with diffuse 23, 48–50
SSc, increased incidence of pulmonary arterial hypertension, skeletal muscle disease, severe cardiac
involvement, and gastrointestinal dysmotility
►► Among SSc patients, anti-U3RNP/fibrillarin antibodies are most commonly found in African American and 48, 49, 51
Latin American patients
Notes: Although some anti-U3RNP/fibrillarin immunoassays are commercially available, technical issues 24
relating to the limited sensitivity of these immunoassays should be taken into consideration.
AC-10 PUNCTATE NUCLEOLAR
►► The AC-10 pattern can be seen in various conditions, including SSc, Raynaud’s phenomenon, SjS, and 52–56
cancer
►► If the AC-10 pattern is observed in the serum of patients with conditions mentioned above, follow-up 54, 55
testing for anti-NOR90(hUBF) antibodies is to be considered; the antigen is included in disease specific
immunoassays (i.e. SSc profile*)
►► While AC-10 is associated with anti-RNApol I antibodies, these antibodies almost always coexist with 52, 53, 57
anti-RNApol III antibodies which reveal the AC-5 pattern; therefore, if SSc is clinically suspected, it
is recommended to perform a follow-up test for anti-RNApol III antibodies (See also AC-5); specific
immunoassays for anti-RNApol I antibodies are currently not commercially available
AC-11 SMOOTH NUCLEAR ENVELOPE
►► The AC-11 pattern is infrequently found in routine autoantibody testing and has been described in 58–60
autoimmune-cytopenias, autoimmune liver diseases, linear scleroderma, APS, and SARD; current
information on clinical associations is based mainly on case reports and small cohorts
►► Antigens recognized include lamins (A, B, C) and LAP-2; specific immunoassays for these autoantibodies 58–60
are currently not commercially available
AC-12 PUNCTATE NUCLEAR ENVELOPE (see online supplementary table S1 for further details)
►► Found in patients with PBC, as well as patients with other autoimmune liver diseases and SARD 61
►► If PBC is clinically suspected, it is recommended to perform a follow-up test for anti-gp210 antibodies; the 62–64
antigen is included in disease specific immunoassays (ie, extended liver profile*)
►► Other antigens recognized include p62 nucleoporin, LBR, and Tpr; specific immunoassays for these 65–68
autoantibodies are currently not commercially available
Continued

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Table 1 Continued
Code AC pattern—clinical relevance Refs
AC-13 PCNA-like (see online supplementary table S1 for further details)
►► The AC-13 pattern has formerly been considered highly specific for SLE, but this specificity is debated 69, 70
►► If SLE is clinically suspected, it is recommended to perform a follow-up test for anti-PCNA antibodies; the 69
antigen is included in several routine ENA profiles
►► Recent studies with antigen-specific immunoassays show clinical associations also with SSc, AIM, RA, HCV, 70–73
and other conditions
AC-14 CENP-F-like
►► The majority of sera exhibiting the AC-14 pattern are from patients with a diversity of neoplastic
conditions (breast, lung, colon, lymphoma, ovary, brain); paradoxically, the frequency of the AC-14 pattern
in patient cohorts with these malignancies is low
►► The AC-14 pattern is also seen in inflammatory conditions (Crohn’s disease, autoimmune liver disease, SjS,
graft-versus-host disease); current information on clinical associations is based mainly on case reports and
series of cases
►► Possible associations only hold if the reactivity to CENP-F is confirmed in an antigen-specific 74–78
immunoassay; current information on clinical associations is based mainly on case reports and series of
cases; specific immunoassays for this autoantibody are currently not commercially available
AC-29 TOPOI-like
►► The AC-29 pattern is highly specific for SSc, in particular with diffuse cutaneous SSc and more aggressive 14, 18, 23
forms of SSc
►► If SSc is clinically suspected, it is recommended to perform a follow-up test for anti-Topoisomerase I 8, 23, 79
(formerly Scl-70) antibodies; the anti-Topoisomerase I antibodies are included in the classification criteria
for SSc and the antigen is included in routine ENA profiles
*Availability of the inflammatory myopathy profile, the SSc profile and the (extended) liver profile may be limited to specialty clinical laboratories.
AIM, autoimmune myopathy; AMA, antimitochondrial antibodies; APS, antiphospholipid syndrome; CENP, centromere-associated protein; DFS, dense fine speckled; DM,
dermatomyositis; ENA, extractable nuclear antigens; HCV, hepatitis C virus; IIFA, indirect immunofluorescence assay; LAP, lamin-associated polypeptide; LBR, lamin B receptor;
LEDGF, lens epithelial derived growth factor; NOR, nucleolus organiser region; NXP, nuclear matrix protein; PBC, primary biliary cholangitis; PCNA, proliferating cell nuclear
antigen; PML, promyelocytic leukaemia; PM/Scl, polymyositis-scleroderma; RA, rheumatoid arthritis; RNApol, RNA polymerase; RNP, ribonucleoprotein; SARD, systemic
autoimmune rheumatic diseases; SLE, systemic lupus erythematosus; SMN, survival of motor neuron; SSc, systemic sclerosis; SjS, Sjögren’s syndrome; TIF, transcription
intermediary factor; TRIM, tripartite motif; Tpr, translocated promoter region; UCTD, undifferentiated connective tissue disease; dsDNA, double stranded DNA; hUBF, human
upstream binding factor.

clinical laboratories is rather inconsistent as shown by external
quality assessments.14 124 125 This is exactly the reason why ICAP
was initiated: the consensus on nomenclature and definitions of
HEp-2 IIFA patterns allows to align pattern description across
laboratories. Also, the integration of computer-aided immu-
nofluorescence microscopy (CAIFM) may further improve the
consistency in pattern assignments.126–131 As such, it is promising
that several companies involved in CAIFM have declared their
intention to accommodate to the ICAP classification. Second,
even apparently healthy individuals may have autoantibodies as
detected by the HEp-2 IIFA. Such autoantibodies, being either
innocent bystander antibodies or predictive antibodies, may
still be present on development of SARD and interfere with
the SARD-related pattern. Interestingly, the pattern best asso-
ciated with apparently healthy individuals is the nuclear dense
fine speckled pattern (AC-2), but this association only holds if
the specificity is confirmed as monospecific for DFS70.20 21 132
Third, the HEp-2 IIFA patterns may slightly differ depending
on the cellular substrate used. For this reason, the ICAP website
contains for each pattern multiple pictures taken from different
brands of HEp-2 slides. Fourth, diseases like systemic lupus
erythematosus and autoimmune inflammatory myopathies may
be associated with distinct autoantibodies, each associated with
a distinct HEp-2 IIFA pattern. If the autoantigens are ill defined,
as is the case, for instance, in autoimmune hepatitis, only the
most prevalent patterns are included. Altogether, it is evident
that, with the exception of the centromere pattern (AC-3), all
patterns are to be confirmed by antigen-specific immunoassay
for a solid association with the respective autoimmune diseases.
While consensus statements have been generated for all 29
HEp-2 IIFA patterns, and it is highly recommended to report
Damoiseaux J, et al. Ann Rheum Dis 2019;78:879–889. doi:10.1136/annrheumdis-2018-214436
patterns,7 11 it is anticipated that laboratories may restrict their
reports to the so-called ‘competent level’ patterns (http://www.​
ANApatterns.​org).133 Although, for instance, the nucleolar
patterns may not be reported as distinct entities (AC-8, AC-9
and AC-10), all three subtypes represent autoantibodies reactive
with antigens associated with systemic sclerosis, either alone or
in combination with autoimmune inflammatory myopathies.
Follow-up testing, therefore, anyhow involves the systemic
sclerosis multiparameter assay including all the relevant auto-
antibodies. Traditionally, only nuclear HEp-2 IIFA patterns
have been considered as a true positive HEp-2 IIFA test, and
this is most likely related to the time-honoured terminology
‘Antinuclear Antibody Test’,12 but it is evident from this report
that even for nuclear HEp-2 IIFA patterns, the clinical associ-
ations are quite diverse. In particular, the nuclear dense fine
speckled pattern (AC-2) seems to have an inverse association
with SARD.9 134 On the other hand, the cytoplasmic HEp-2 IIFA
patterns, and to a lesser extent the mitotic patterns, are also
clinically relevant and may demand dedicated follow-up testing
in daily clinical practice. Therefore, the ICAP executive board
advocates that information on HEp-2 IIFA patterns should be
reported to the clinician and should also be incorporated in diag-
nostic and classification criteria instead of the simple assignment
‘ANA-positive’.135
Although the HEp-2 IIFA has been considered the gold stan-
dard for autoantibody detection in SARD,3 the limitations of
this assay are understood.136–138 Indeed, up to 35% of healthy
controls may be positive if a screening dilution of 1/40 is used.139
Therefore, in the EASI/IUIS recommendations, it is advocated
that each laboratory verifies that the screening dilution is
defined by a cut-off set at the 95th percentile.7 However, by
883
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Table 2 Cytoplasmic HEp-2 IIFA patterns
Code AC pattern–—clinical relevance Refs
AC-15 FIBRILLAR LINEAR (see online supplementary table S2 for further details)
►► Found in patients with AIH type 1, chronic HCV infection, and celiac disease 17
(IgA isotype); rare in SARD
►► If AIH type 1 is clinically suspected, it is recommended to confirm reactivity 17,80
with smooth muscle antibodies (IgG isotype), typically detected by IIFA on
rodent tissue (liver, stomach, kidney); anti-smooth muscle antibodies are
included in the international criteria for AIH type 1
►► F-actin is the main target antigen of anti-smooth muscle antibodies in 81–83
AIH type 1; autoantibodies to F-actin are of more clinical importance than
antibodies to G-actin
Notes: Although anti-F-actin immunoassays are commercially available, technical
issues relating to the sensitivity of these immunoassays should be taken into
consideration.
AC-16 FIBRILLAR FILAMENTOUS (see online supplementary table S2 for further details)
►► Found in various diseases, but AC-16 is not typically found in SARD
►► Antigens recognized include cytokeratins 8, 18, & 19, tubulin, and
vimentin; specific immunoassays for these autoantibodies are currently not
commercially available
AC-17 FIBRILLAR SEGMENTAL (see online supplementary table S2 for further details)
►► Found very infrequently in a routine serology diagnostic setting
►► Antigens recognized include α-Actinin and Vinculin; specific immunoassays
for these autoantibodies are currently not commercially available
AC-18 DISCRETE DOTS (see online supplementary table S2 for further details)
►► Autoantibodies revealing the AC-18 pattern have been reported in distinct 84
SARD and in a variety of other diseases; their prevalence in unselected or
specified disease cohorts has not been thoroughly studied
►► Antigens recognized include GW-body (Processing or P body) antigens (Ge-
1/Hedls, GW182, and Su/Ago2) and endosomal antigens (EEA1, CLIP-170,
GRASP-1, and LBPA); specific immunoassays for these autoantibodies are
currently not commercially available
Notes: Autoantibodies to GW-bodies and endosomes may yield slightly different 84, 85
HEp-2 IIFA patterns.
AC-19 DENSE FINE SPECKLED (see online supplementary table S2 for further details)
►► Found in patients with SLE and the anti-synthetase syndrome (a subset of 33, 86, 87
AIM), interstitial lung disease, polyarthritis, Raynaud’s phenomenon, and
mechanic’s hands; these features may occur in various combinations or as
an isolated manifestation, especially interstitial lung disease
►► If SLE is clinically suspected, follow-up tests for antibodies to ribosomal P
phosphoproteins (P0, P1, P2, C22 RibP peptide) are recommended; these
antigens may be included in the routine ENA profile
►► Anti-RibP antibodies have been associated in some studies with 86, 88, 89
neuropsychiatric lupus, and in childhood-onset SLE with autoimmune
hemolytic anemia
►► If AIM, in particular the anti-synthetase syndrome, is suspected, it 26, 33
is recommended to perform follow-up tests for antibodies to tRNA
synthetases; antigens are included in disease specific immunoassays (ie,
inflammatory myopathy profile*)
►► If AIM, in particular necrotizing myopathy, is suspected, it is recommended 26
to perform follow-up tests for anti-SRP antibodies; the antigen is included in
disease specific immunoassays (ie, inflammatory myopathy profile*)
Notes: The fine distinction between AC-19 and -20 may depend on HEp-2
substrates and/or antibody concentration; antibodies to both RibP as well as
tRNA synthetases may be undetected in HEp-2 IIFA-screening.
AC-20 FINE SPECKLED
►► Found in patients with the anti-synthetase syndrome (a subset of AIM), 33, 90
interstitial lung disease, polyarthritis, Raynaud’s phenomenon, and
mechanic’s hands; these features may occur in various combinations or as
an isolated manifestation, especially interstitial lung disease
►► Autoantibodies associated with the AC-20 pattern are primarily reported 91, 92
for the anti-Jo-1 antibody, which recognizes histidyl-tRNA synthetase; since
AC-20 is not specific for Jo-1, it is recommended to perform a follow-up test
for anti-Jo-1 antibodies; the antigen is included in the routine ENA profile,
as well as in disease specific immunoassays (i.e., inflammatory myopathy
profile*); the anti-Jo-1 antibodies are included in the classification criteria
for AIM
Notes: The fine distinction between AC-19 and -20 may depend on HEp-2
substrates and/or antibody concentration; antibodies to Jo-1 may be undetected
in HEp-2 IIFA-screening.
Continued

884 Damoiseaux J, et al. Ann Rheum Dis 2019;78:879–889. doi:10.1136/annrheumdis-2018-214436

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Table 2 Continued
Code AC pattern–—clinical relevance Refs
AC-21 RETICULAR/AMA
►► Commonly found in PBC, but also detected in SSc, including PBC-SSc 93–97
overlap syndrome and PBC-SjS overlap syndrome
►► If PBC is clinically suspected it is recommended to perform a follow-up 93, 94
test for AMA, historically detected by IIFA on rodent tissue (liver, stomach,
kidney); these autoantibodies are primarily directed to the PDH complex,
and in particular the E2-subunit (PDH-E2); the antigen is included in disease
specific immunoassays (i.e., liver profile*) as well as in some routine ENA
profiles
►► Additional antigens recognized include the E1α and E1β subunits of PDH, 93, 94
the E3-binding protein of PDH, and the 2-OGDC; these antigens are only
included in extended disease specific immunoassays (i.e., extended liver
profile*)
AC-22 POLAR/GOLGI-like (see online supplementary table S2 for further details)
►► Found in small numbers of patients with a variety of conditions
►► Antigens recognized include giantin/macrogolgin and distinct golgin 85
molecules; specific immunoassays to detect autoantibodies directed to
specific Golgi antigens are currently not commercially available
AC-23 RODS and RINGS (see (online supplementary table S2 for further details)
►► Most commonly found in HCV patients who have been treated with 98–101
pegylated interferon-α/ribavirin combination therapy, but autoantibodies
revealing the AC-23 patterns were undetected prior to treatment; as the use
of interferon-α/ribavirin in HCV treatment is decreasing, the frequency and
clinical associations of the AC-23 pattern may change
►► Specific immunoassays to detect autoantibodies directed to specific Rods
and Rings antigens, for instance IMPDH2, are not commercially available
Note: Presence of the AC-23 pattern depends on the HEp-2 cell substrate.
*Availability of the inflammatory myopathy profile, the SSc profile and the (extended) liver profile may be limited to specialty clinical laboratories.
AIH, autoimmune hepatitis; AIM, autoimmune myopathy; AMA, anti-mitochondrial antibodies; APS, antiphospholipid syndrome; Ago, argonaute protein; CENP, centromere-associated protein;
CLIP, class II-associated invariant chain peptide; DFS, dense fine speckled; DM, dermatomyositis; EEA, early endosome antigen; ENA, extractable nuclear antigens; HCV, hepatitis C virus; IFA,
indirect immunofluorescence assay; LAP, lamin-associated polypeptide; LBR, lamin B receptor; LEDGF, lens epithelial derived growth factor; NOR, nucleolus organizer region; NXP, nuclear matrix
protein; PBC, primary biliary cholangitis; PCNA, proliferating cell nuclear antigen; PML, promyelocytic leukaemia; PM/Scl, polymyositis-scleroderma; RA, rheumatoid arthritis; RNApol, ribonucleic
acid polymerase; RNP, ribonucleoprotein; SARD, systemic autoimmune rheumatic diseases; SLE, systemic lupus erythematosus; SMN, survival of motor neuron; SRP, signal recognition protein; SSc,
systemic sclerosis; SjS, Sjögren’s syndrome; TIF, transcription intermediary factor; TRIM, tripartite motif; Tpr, translocated promoter region; dsDNA, double stranded deoxyribonucleic acid; hUBF,
human upstream binding factor; tRNA, transfer ribonucleic acid.

taking into account that the HEp-2 IIFA nowadays is ordered
by a wide spectrum of clinical disciplines,1 the number of clin-
ically unexpected positive results, that is, positive test results
with no clinical evidence of an associated autoimmune disease,
is ever increasing and may even equal the likelihood of a clini-
cally true-positive result.140 141 A study performed in a commu-
nity setting concluded that many patients with a positive ANA
test are incorrectly given a diagnosis of systemic lupus eryther-
matosus and sometimes even treated with toxic medications.142
These arguments are used to introduce a gating strategy in order
to restrict test-ordering to those cases that have a sufficiently
high pretest probability for having a SARD. However, it can also
be argued that patients with a low pretest probability should be
tested using the HEp-2 IIFA in order to prevent true cases, espe-
cially those with very early disease manifestations, from being
missed. This is a paradigm shift to disease prediction and preven-
tion.143 In this strategy, the HEp-2 IIFA could be integrated in
multianalyte ‘omic’ profiles for case finding and establishing an
early diagnosis and preventing severe complications.143 144 Obvi-
ously, it is anticipated that the added value of the HEp-2 IIFA in
this approach can be increased by incorporating information on
both patterns as well as titres in combination with well-directed
advices on follow-up testing.
Although the current consensus on the clinical relevance of
HEp-2 IIFA patterns has come across after extensive discussion
and debate within the ICAP executive board as well as with the
workshop participants, the information provided is not based
on a systematic review or meta-analysis of the existing litera-
ture. Because of the short history of ICAP, being founded in
2014, inclusion of older literature might have been hampered
Damoiseaux J, et al. Ann Rheum Dis 2019;78:879–889. doi:10.1136/annrheumdis-2018-214436
by potential differences in pattern nomenclature and defi-
nitions. For instance, the nuclear dense fine speckled (AC-2)
and topo I-like (AC-29) patterns were previously often consid-
ered homogeneous, speckled or even mixed patterns. The
centromere pattern (AC-3) or the cytoplasmic reticular/AMA
(AC-21) patterns, on the other hand, are examples that prob-
ably have been less prone to change in pattern definition over
time. The universal use of the ICAP nomenclature and pattern
definitions, both in daily clinical practice as well as in the scien-
tific literature, may enable systematic reviews in the future, and
may well fine-tune current consensus based on expert opinions
only.
In conclusion, the consensus statements on clinical relevance
should be readily available to clinicians and this will enable
further harmonisation of test-result interpretation with respect
to HEp-2 IIFA patterns. Obviously, clinicians should be aware
of the clinical suspicion for the respective patient, and therefore
should order specific tests accordingly, also taking into account
the anticipation of prevalence of HEp-2 IIFA negative (AC-0)13
results in SARD. The information on clinical relevance of HEp-2
IIFA patterns is intended to support the decision strategy of the
clinician. Information presented in the online supplementary
tables 1–3 is primarily intended to be used for complex cases
in the consultation of the laboratory specialist by the clinician.
Depending on various jurisdictional regulations, follow-up
testing can be automated in predefined algorithms which even-
tually will shorten the diagnostic delay. Eventually, appropriate
integration of HEp-2 IIFA pattern information may help to
better define disease criteria and even enable a paradigm shift in
the pretest probability paradox.
885
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7
Medicine, Health Sciences Centre, Calgary, Alberta, Canada
Table 3 Mitotic HEp-2 IIFA patterns 8
Department of Immunology and Rheumatology, Hospital General de Occidente,
Code AC pattern—clinical relevance Refs Guadalajara, Mexico
9
Rheumatology Unit, Clinical Department of General Internal Medicine, Innsbruck
AC-24 CENTROSOME (see online supplementary table 3 for further Medical University, Innsbruck, Austria
details) 10
Department of Internal Medicine II, Medical University of Innsbruck, Innsbruck,
►► The AC-24 pattern has low positive predictive value for Austria
any disease 11
Pontificia Universidade Catolica de Goias, Goiania, Brazil
12
►► Within the spectrum of the SARD, the AC-24 pattern 102–105 Department of Rheumatology and Clinical Immunology, Kyoto University Graduate
is found in patients with Raynaud’s phenomenon, school of Medicine, Kyoto, Japan
localized scleroderma, SSc, SLE and RA, either alone or in 13
Brazilian Society of Autoimmunity, Porto Alegre, Brazil
combination with other SSc-associated antibodies; 14
Department of Clinical Nursing, University of Occupational and Environmental
►► Antigens recognized include α-enolase, γ-enolase, 104, 106–108 Health, Kitakyushu, Japan
15
ninein, Cep-250, Mob1, PCM-1/2, pericentrin; specific Department of Oral Biology, University of Florida, Gainesville, Florida, USA
immunoassays for these autoantibodies are currently not
commercially available
Acknowledgements We thank all the workshop participants for their constructive
comments and fruitful discussions.
AC-25 SPINDLE FIBERS (see online supplementary table 3 for further
details) Contributors All authors actively participated in the respective workshops in
►► The AC-25 pattern has low positive predictive value for 109
Kyoto and Dresden. They also participated in the discussions of the executive ICAP
any disease committee. The draft of the manuscript was made by JD and was commented on by
all authors. Final discussions have taken place at the international autoimmunity
►► Found very infrequently in a routine serology diagnostic 109
meeting in Lisbon. Required amendments were made by JD and approved by all
setting
authors.
►► Antigen recognized includes HsEg5; specific 110, 111
immunoassays for this autoantibody, or other spindle Funding The authors have not declared a specific grant for this research from any
fiber targets, are currently not commercially available funding agency in the public, commercial or not-for-profit sectors.
AC-26 NuMA-like Competing interests The ICAP committee is funded by unrestricted educational
►► Approximately one-half of the patients with the AC-26 109, 111–114 grants by several in vitro diagnostics companies (for details see www.​anapatterns.​
pattern have clinical features of a SARD (SjS, SLE, UCTD, org/​sponsors.​php). JD has received lecture fees from Euroimmun and Thermo Fisher.
limited SSc, or RA); the AC-26 pattern is also observed in MJF is a consultant to Inova Diagnostics and Werfen International; none of the other
patients with organ-specific autoimmune diseases and authors declare any competing interest.
less frequently in non-autoimmune conditions, especially
Patient consent for publication Not required.
when in low titer
►► Found very infrequently in a routine serology diagnostic 109 Provenance and peer review Not commissioned; externally peer reviewed.
setting Data sharing statement No additional data are available.
►► Antigens recognized include NuMA, centrophilin, SP-H 115
Open access This is an open access article distributed in accordance with the
antigen and NMP-22; specific immunoassays for these
Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which
autoantibodies are currently not commercially available
permits others to distribute, remix, adapt, build upon this work non-commercially,
AC-27 INTERCELLULAR BRIDGE (see online supplementary table 3 and license their derivative works on different terms, provided the original work is
for further details) properly cited, appropriate credit is given, any changes made indicated, and the use
►► The AC-27 pattern has low positive predictive value for 116 is non-commercial. See: http://​creativecommons.​org/​licenses/​by-​nc/​4.​0/.
any disease
►► Found very infrequently in a routine serology diagnostic 117
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